One of the early steps in malignant progression is tumor angiogenesis, supplying nutrients to the developing tumor and possibly providing routes for tumor cell escape. In numerous transplant and transgenic models, subsets of BMDCs were shown to deliver MMP-9 to mediate the angiogenic switch and frequently, the MMP-9-delivering BMDCs were identified as monocytes/macrophages. A few studies indicated that neutrophils might serve as an alternative BMDC type since they also express MMP-9 and have been associated, although transiently, with developing tumors. However, inflammatory neutrophils, with their only clear role in acute infection and injury, have generally been overlooked as contributors to tumor progression. We have recently demonstrated that neutrophils produce a unique, TIMP-free form of proMMP-9, making it a potent angiogenic agent. In light of this evidence and that neutrophils infiltrate tumor-stromal tissue and rapidly release their prestored, highly-angiogenic TIMP-free MMP-9, we would challenge the existing paradigms that monocyte-like cells are exclusive inducers of the angiogenic switch. We propose as an overall hypothesis that the initial delivery of neutrophils and their unique MMP-9 is a critical and targetable early event in tumor progression. To test this hypothesis we propose the following Specific Aims: Aim 1: To Directly Compare the Angiogenic Capabilities of Inflammatory Neutrophils and Their Unique TIMP-Free MMP-9 with Differentiating Monocytes and Their Secreted MMP-9. Comparisons will include kinetic studies of MMP-9 release/production by the different myeloid cell types, influx of MMP-9-delivering inflammatory cells into angiogenic and tumor tissues, and quantifying the in vivo angiogenic activity of MMP-9 delivered by inflammatory neutrophils versus monocytes. Aim 2: To Elucidate the Biochemical Mechanism(s) which Determine the High Pro-Angiogenic and Pro-Tumor Progression Potentials of Neutrophil MMP-9. The inducers of neutrophil influx into tumor tissues will be quantified. The mechanism of activation of the TIMP-free neutrophil MMP-9 zymogen will be determined, which in vivo has never been documented. The plasminogen/plasmin serine protease cascade will be examined mechanistically for partnering with activated MMP-9 during angiogenic induction. The in vivo substrates and effector molecules of activated neutrophil MMP-9 will be probed by new proteomic approaches. Aim 3: To Validate That the Pro-Angiogenic Activity of Neutrophil MMP-9 is Causally Linked to Tumor Dissemination via Neovascular Conduits. The specific reduction or stimulation of neutrophil MMP-9-mediated angiogenesis will be related directly to the levels of tumor cell intravasation and confirmed with in vivo rescue experiments using viable neutrophils and/or purified neutrophil MMP-9. The results will be then validated in a number of murine models of angiogenesis and metastasis including orthotopic transplants and transgenic models focusing on the human disease-like TRAMP model. PUBLIC HEALTH RELEVANCE: Deaths from cancer occur mainly because tumor cells spread from the primary tumor site to other vital organs and tissues through the new angiogenic vasculature of the primary tumor. One of the non-tumor cell types of the host that can actively contribute to the induction of new angiogenic blood vessels feeding the tumor is the inflammatory neutrophil, which can import molecules that directly induce the formation of new blood vessels and do so early on in tumor progression. The goal of the proposed research is to biochemically and mechanistically examine the pro-malignant role of the unique neutrophil enzyme, MMP-9, one of the few molecules released by neutrophils that is a potent inducer of angiogenesis and likely an inducer of tumor metastasis and thus could be therapeutically targeted in cancers associated with inflammatory neutrophil infiltration.